Viral vectors for gene-directed enzyme prodrug therapy

Conventional cancer treatments are often hampered by a lack of tumour selectivity, resulting in toxicity to healthy tissue. Gene-directed enzyme prodrug therapy (GDEPT) is a suicide gene therapy approach that aims to improve the selectivity of chemotherapy by enabling cancer cells to convert non-cytotoxic prodrugs to cytotoxic drugs. Many enzyme/prodrug systems have been described, some of which have already been tested in clinical trials. A key component of GDEPT is a foreign enzyme that is expressed selectively at the tumour site where it converts the prodrug into the cytotoxic agent. The gene encoding the prodrug-activating enzyme needs to be expressed selectively and efficiently in tumour cells in order to spare normal tissue from damage. Substantial efforts have been made to develop gene therapy vectors that are capable of targeting cancer cells. A large number of gene delivery systems have been described for GDEPT: Viral vectors are the most advanced. They include replication-deficient and replication-selective (oncolytic) viruses. Recent advances in engineering viruses for GDEPT are reviewed in this article and data from both preclinical studies and clinical trials are discussed.     

细胞色素P450与肿瘤的基因治疗

利用细胞色素P450治疗肿瘤是一种新的基因治疗方法,对提高肿瘤化疗的安全性和有效性有非常大的潜力。这种方法的首要目标就是选择性地使细胞色素P450在肿瘤细胞内超量表达。P450酶系降解的前药大部分在肿瘤内活化,提高了肿瘤细胞内药物的相对浓度,减小药物对其它组织的细胞毒性。本文概述了可被细胞色素P450降解的用于肿瘤治疗的前药以及重组细胞色素P450在基因治疗中的应用。     

Viral based gene therapy for prostate cancer

In the last few years, significant advances in gene therapy have been made as a result of advances in many areas of molecular and cell biology, including the improvement of both viral and nonviral gene delivery systems, discovery of new therapeutic genes, better understanding of mechanism of disease progression, exploration of tissue specific promoter, receptor- and antibody-mediated targeting delivery, and development of better prodrug enzyme/prodrug systems. In this article, viral based gene therapy for prostate cancer will be reviewed and discussed. The areas of emphasis in this review are: choice of viral vectors, comparison of delivery routes, development of prostate-targeted viruses, choice of therapeutic genes and strategies including corrective gene therapy (tumor suppressor gene and anti-oncogene gene approaches), suicide gene therapy, programmed cell death therapy, immunomodulation therapy, and conditional oncolytic virus approach. Among them, several examples will be discussed in detail for the scientific basis and therapeutic applications. In addition, prostate cancer gene therapy clinical trials, unresolved problems and future directions in this field will also be described.     

Gene Therapy for Brain Tumors

Gene therapy has opened new doors for treatment of neoplastic diseases. This new approach seems very attractive, especially for glioblastomas, since treatment of these brain tumors has failed using conventional therapy regimens. Many different modes of gene therapy for brain tumors have been tested in culture and in vivo. Many of these approaches are based on previously established anti-neoplastic principles, like prodrug activating enzymes, inhibition of tumor neovascularization, and enhancement of the normally weak anti-tumor immune response. Delivery of genes to tumor cells has been mediated by a number of viral and synthetic vectors. The most widely used paradigm is based on the activation of ganciclovir to a cytotoxic compound by a viral enzyme, thymidine kinase, which is expressed by tumor cells, after the gene has been introduced by a retroviral vector. This paradigm has proven to be a potent therapy with minimal side effects in several rodent brain tumor models, and has proceeded to phase 1 clinical trials. In this review, current gene therapy strategies and vector systems for treatment of brain tumors will be described and discussed in light of further developments needed to make this new treatment modality clinically efficacious.     

In situ gene therapy for prostate cancer

The incidence of prostate cancer has dramatically increased worldwide in the past decade, with mortality rates also increasing in many countries. Once prostate cancer is diagnosed, it is important to rapidly begin a treatment regimen that is either potentially curative or impedes disease progression. When the disease is confined to the prostate, it can be cured by radical prostatectomy or irradiation therapy. However, there are no curative therapies for locally advanced, recurrent, or metastatic diseases. Clearly, new therapies are needed for these patients. Gene therapy may provide additional therapeutic options with the potential to affect both localized and metastatic disease. Virus-mediated transduction of the herpes simplex virus thymidine kinase (HSV-tk) gene transfer, followed by a course of the prodrug ganciclovir (GCV), so-called suicide gene therapy, has been demonstrated by several investigators. The present in situ gene therapy clinical trial for human prostate cancer demonstrated safety, clinical efficacy, and biological effects of antitumor activity. HSV-tk clinical trials for prostate cancer are also ongoing in Japan, the Netherlands, and Mexico. Currently, numerous preclinical studies have reported immunomodulatory cytokine gene therapy, such as interleukin-2, interleukin-12, B7-1 (CD80), B7-2 (CD86) and granulocyte-macrophage colony-stimulating factor. Several clinical studies have been approved that potentially will show that these immunomodulatory gene therapies may generate an effective local and systemic antitumor activity and that should provide options for patients with prostate cancer. We review the multiple issues involved in current in situ gene therapy (gene/immunotherapy), its outcome, and future directions for patients with prostate cancer.     

A novel technique to monitor carboxypeptidase G2 expression in suicide gene therapy using 19F magnetic resonance spectroscopy

Development and evaluation of new anticancer drugs are expedited when minimally invasive biomarkers of pharmacokinetic and pharmacodynamic behaviour are available. Gene‐directed enzyme prodrug therapy (GDEPT) is a suicide gene therapy in which the anticancer drug is activated in the tumor by an exogenous enzyme previously targeted by a vector carrying the gene. GDEPT has been evaluated in various clinical trials using several enzyme/prodrug combinations. The key processes to be monitored in GDEPT are gene delivery and expression, as well as prodrug delivery and activation. {4‐[bis(2‐chloroethyl)amino]‐3,5‐difluorobenzoyl}‐L‐glutamic acid, a prodrug for the GDEPT enzyme carboxypeptidase‐G2 (CPG2; K_m = 1.71 µM; k_cat = 732 s^?1), was measured with ¹⁹F magnetic resonance spectroscopy (MRS). The 1 ppm chemical shift separation found between the signals of prodrug and activated drug (4‐[bis(2‐chloroethyl)amino]‐3,5‐difluorobenzoic acid) is sufficient for the detection of prodrug activation in vivo. However, these compounds hydrolyze rapidly, and protein binding broadens the MR signals. A new CPG2 substrate was designed with hydroxyethyl instead of chloroethyl groups (K_m = 3.5 µM, k_cat = 747 s^?1). This substrate is nontoxic and stable in solution, has a narrow MRS resonance in the presence of bovine and foetal bovine albumin, and exhibits a 1.1 ppm change in chemical shift upon cleavage by CPG2. In cells transfected to express CPG2 in the cytoplasm (MDA MB 361 breast carcinoma cells and WiDr colon cancer cells), well‐resolved ¹⁹F MRS signals were observed from clinically relevant concentrations of the new substrate and its nontoxic product. The MRS conversion half‐life (470 min) agreed with that measured by HPLC (500 min). This substrate is, therefore, suitable for evaluating gene delivery and expression prior to administration of the therapeutic agent. Copyright © 2009 John Wiley & Sons, Ltd.     

Lentivirus-mediated expression of Drosophila melanogaster deoxyribonucleoside kinase driven by the hTERT promoter combined with gemcitabine: a potential strategy for cancer therapy

In contrast to other enzymes, Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) has a broad substrate specificity and high catalytic rate when transferred in human cells. This makes it a promising therapeutic agent when administered together with several cytotoxic nucleoside analogs, such as gemcitabine 2',2'-difluoro-deoxycytidine (dFdC). Therefore, lentiviral vectors, which potentially allow stable long-term transgene expression, are good candidates for gene delivery vehicles. In the present study, we successfully developed a lentivirus-mediated transgene expression system of Dm-dNK under the control of hTERT promoter against the breast cancer cell line (Bcap37), the gastric cancer cell line (SGC7901) and the normal fibroblast cell line (WI-38). Moreover, we also analyzed its targeted cytotoxicity in vitro with treatment of the prodrug dFdC. Bcap37 tumor growth was inhibited in nude mice. Both cancer cell lines exhibited apparent cytotoxicity when infected with recombinant lentivirus constructs expressing Dm-dNK. In contrast, lentivirus-infected WI-38 cells exhibited less cytotoxicity. These data suggested that Dm-dNK was sensitive to dFdC, and it resulted in synergistic growth inhibition and apoptosis induction in vitro. In addition, Lenti-hTERT-dNK/dFdC also suppressed tumor growth in vivo. Our results suggest that the Lenti-hTERT-dNK/dFdC system is a safe and feasible treatment strategy in the development of suicide gene therapy.     

Immuno-isolation in cancer gene therapy

The implantation of genetically-modified non-autologous cells in immuno-protected microcapsules is an alternative to ex vivo gene therapy. Such cells delivering a recombinant therapeutic product are isolated from the host's immune system by being encapsulated within permselective microcapsules. This approach has been successful in pre-clinical animal studies involving delivery of hormone or enzymes to treat dwarfism, lysosomal storage disease, or hemophilia B. Recently, this platform technology has shown promise in the treatment for more complex diseases such as cancer. One of the earliest strategy was to augment the chemotherapeutic effect of a prodrug by implanting encapsulated cells that can metabolise prodrugs into cytotoxic products in close proximity to the cancer cells. More recent approaches include enhancing tumor cell death through immunotherapy, or suppressing tumor cell proliferation through anti-angiogenesis. These can be achieved by delivering single molecules of cytokines or angiostatin, respectively, by implanting microencapsulated cells engineered to secrete these recombinant products. Recent refinements of these approaches include genetic fusion of cytokines or angiostatin to additional functional groups with tumor targeting or tumor cell killing properties, thus enhancing the potency of the recombinant products. Furthermore, a COMBO strategy of implanting microencapsulated cells to deliver multiple products targeted to diverse pathways in tumor suppression also showed much promise. This review will summarise the application of microencapsulation of genetically-modified cells to cancer treatment in animal models, the efficacy of such approaches, and how these studies have led to better understanding of the biology of cancer treatment. The flexibility of this modular system involving molecular engineering, cellular genetic modification, and polymer chemistry provides potentially a huge range of application modalities, and a tremendous multi-disciplinary challenge for the future.     

Suicide gene therapy mediated by the Herpes Simplex virus thymidine kinase gene/Ganciclovir system: fifteen years of application

Gene-directed enzyme prodrug therapy (GDEPT) is a two step therapeutic approach for cancer gene therapy. In the first step, the transgene is delivered into the tumor and expressed. In the second step a prodrug is administered and is selectively activated by the expressed enzyme. The first GDEPT system described was the thymidine kinase gene of the Herpes Simplex virus (HSVtk) in combination with the prodrug Ganciclovir (GCV). A large number of experiments have been performed with this system, in different types of tumors and initial studies in animal models were very promising. This encouraged investigators to move into clinical trials although poor results have been obtained so far. A large effort has been made with numerous different strategies to enhance HSVtk/GCV efficacy in cellular and in vivo models and very strong cytotoxic effects have been obtained. The present review describes the current state of preclinical research and summarizes the results of the clinical trials undertaken.     

The use of folate-PEG-grafted-hybranched-PEI nonviral vector for the inhibition of glioma growth in the rat

Combined treatment using nonviral agent-mediated enzyme/prodrug therapy and immunotherapy had been proposed as a powerful alternative method of cancer therapy. The present study was aimed to evaluate the cytotoxicity in vitro and the therapeutic efficacy in vivo when the cytosine deaminase/5-fluorocytosine (CD/5-FC) and TNF-related apoptosis-inducing ligand (TRAIL) genes were jointly used against rat C6 glioma cells. The potency of the FA-PEG-PEI used as a nonviral vector was tested in the FR-expressed C6 glioma cells and Wistar rats. The C6 glioma cells and animal model were treated by the combined application of FA-PEG-PEI/pCD/5-FC and FA-PEG-PEI/pTRAIL. The antitumor effect was evaluated by survival assays and tumor volume. This study revealed a significant increase of cytotoxicity in vitro following the combined application of FA-PEG-PEI/pCD/5-FC and FA-PEG-PEI/pTRAIL treatments in C6 glioma cells. Animal studies showed a significant growth inhibition of the C6 glioma xenografts using the combined treatment. These results demonstrated that the combined treatment generated additive cytotoxic effect in C6 glioma cells in both in vitro and in vivo conditions, and indicated that such treatment method using both enzyme/prodrug therapy and TRAIL immunotherapy might be a promising therapeutic strategy in treating glioma.     

Influence of the bystander effect on HSV-tk/GCV gene therapy. A review

Despite the development of new therapeutic strategies, cancer remains incurable in most patients with advanced disease. A recent potential improvement in therapeutic strategies is the concept of suicide gene therapy. After transfection with a suicide gene, cells can convert a harmless prodrug into its toxic metabolite, resulting in selective elimination of these cells. One of the most frequently studied therapeutic strategies is based on transfection with the herpes simplex virus thymidine kinase gene (HSV-tk), followed by ganciclovir administration. Despite promising results in vitro and in vivo, the antitumor effect in clinical trials remains poor, due to very low transfection efficiency. However, high percentages of transfected cells are not mandatory for complete eradication of a tumor in vivo. Transfected tumor cells appear to be capable of inducing the death of neighboring untransfected cells. This cell kill is called the "bystander effect". Various attempts have been made to increase this effect. A substantial bystander effect could overcome the limitations of low transfection efficiency and result in an enhanced and possibly clinically worthwhile antitumor effect in patients. This review is focused on the HSV-tk/GCV system and gives an overview of current knowledge on the bystander effect in vitro and in vivo. In addition, theories concerning its mechanisms and possible approaches to augment this effect are discussed. Finally, we give an overview of clinical trials using suicide gene therapy.     

Prodrugs for Gene-Directed Enzyme-Prodrug Therapy (Suicide Gene Therapy)

This review focuses on the prodrugs used in suicide gene therapy.These prodrugs need to satisfy a number of criteria. They must be efficient and selective substrates for the activating enzyme, and be metabolized to potent cytotoxins preferably able to kill cells at all stages of the cell cycle. Both prodrugs and their activated species should have good distributive properties, sothat the resulting bystander effects can maximize the effectiveness of the therapy, since gene transduction efficiencies are generally low. A total of 42 prodrugs explored for use in suicide gene therapy with 12 different enzymes are discussed, particularly in terms of their physiocochemical properties. An important parameter in determining bystander effects generated by passive diffusion is the lipophilicity of the activated form, a property conveniently compared by diffusion coefficients (log P for nonionizable compounds and log D₇ for compounds containing an ionizable centre). Many of the early antimetabolite-based prodrugs provide very polar activated forms that have limited abilities to diffuse across cell membranes, and rely on gap junctions between cells for their bystander effects.Several later studies have shown that more lipophilic, neutral compounds have superior diffusion-based bystander effects. Prodrugs of DNA alkylating agents, that are less cell cycle-specific than antimetabolites and more effective againstnoncycling tumor cells, appear in general to be more active prodrugs, requiring less prolonged dosing schedules to be effective. It is expected that continued studies to optimize the bystander effects and other properties of prodrugs and theactivated species they generate will contribute to improvements in the effectiveness of suicide gene therapy.     

Developments with targeted enzymes in cancer therapy.

Cancer therapy based on the delivery of enzymes to tumour sites has advanced in several directions since antibody-directed enzyme/prodrug therapy was first described. It has been shown that methoxypolyethylene glycol (MPEG) can be used to deliver enzyme to a variety of solid tumours. MPEG-enzyme conjugates show reduced immunogenicity and may allow repeated treatment with enzymes of bacterial origin. Enzyme delivery to tumours by polymers can be used to convert a low toxicity prodrug to a potent cytotoxic agent. An example of such a prodrug is CB1954, which can be activated by a human enzyme in the presence of a cosubstrate. Tumour-located enzymes can also be used in conjunction with a combination of antimetabolites and rescue agents. The rescue agent protects normal tissue but is degraded at cancer sites by the enzyme, thus deprotecting the tumour and allowing prolonged antimetabolite action.     

Plasmidic CpG sequences induce tumor microenvironment modifications in a rat liver metastasis model

Bacterial DNA contains unmethylated cytosine-phosphate-guanine (CpG) motifs which are recognized by mammalian immune cells as a danger signal indicating an infection. These immunostimulatory properties led to the use of oligodeoxynucleotides bearing CpG motifs (CpG-ODN) for cancer treatment in preclinical and clinical studies. Although naked DNA administration presently represents 18% of the gene therapy clinical trials worldwide, most of the work regarding the effects of unmethylated CpG sequences was performed using CpG-ODN. In the present study, we analyzed early induced tumor microenvironment modifications in a rat liver metastasis model after intratumoral injection of a plasmid used in suicide gene therapy. We first showed that plasmidic CpG motifs were active, i.e. able to induce IFN-gamma secretion by rat splenocytes. Then, we compared tumor-infiltrating immune cells 24 h after injection of native or SssI-treated plasmid, in which immunostimulatory CpG motifs have been inactivated by methylation. The presence of active plasmidic CpG sequences within the tumor was associated with a decrease in the number of tumor-infiltrating conventional dendritic cells and an upregulation of the CCR7 chemokine receptor responsible for lymph node homing. We also observed an increase in plasmacytoid dendritic cells and natural killer cell infiltration within the tumors as well as an increased mRNA expression of three cytokines/chemokines (IL-1beta, IL-10 and IL-18). These data suggest that, although suicide plasmid injection without prodrug treatment is not sufficient to observe a therapeutic effect, the presence of plasmidic CpG motifs within the tumor induces the recruitment and activation of the immune cells involved in antitumor response. These early cellular and molecular events should facilitate the induction of the immune response against tumor antigens released after in situ drug production.     

Gene therapy for colorectal cancer: therapeutic potential

Colorectal cancer is a leading cause of cancer mortality in Western countries. Gene therapy has been proposed as a potential novel treatment modality for colorectal cancer, but it is still in an early stage of development. The preclinical data have been promising and numerous clinical trials are underway. This brief review aims to summarise the current status of clinical trials of different gene therapy strategies, including immune stimulation, mutant gene correction, prodrug activation and oncolytic virus therapy, for patients with colorectal cancer. Data from phase I trials have proven the safety of the reagents but have not yet demonstrated significant therapeutic benefit. In order to achieve this and extend the scope of the treatment, continuing efforts should be made to improve the antitumour potency, efficiency of gene delivery and accuracy of gene targeting.     

Selective chemotherapeutic strategies using catalytic antibodies: a common pro-moiety for antibody-directed abzyme prodrug therapy

Prodrug activation by catalytic antibodies (abzymes) conjugated with anti-tumor antibodies, called antibody-directed abzyme prodrug therapy (ADAPT), has been proposed as a strategy for site-specific drug delivery systems for anti-tumor drugs. The delivery of abzymes is achieved by making a bi-specific antibody with a monovalent catalytic antibody and a monovalent binding antibody. To achieve ADAPT, we focused on specific requirements for prodrugs and catalytic antibodies, the stability of the prodrugs against natural enzymes, and the applicability of abzymes for a wide range of prodrugs. Attention was paid to the design of a pro-moiety rather than a parent drug. As a common pro-moiety, we chose vitamin B(6), because the bulky vitamin B(6) esters are relatively stable against hydrolytic enzymes in serum. We have generated catalytic antibodies by immunization of a vitamin B(6) phosphonate transition state analog. The elicited antibodies were found to hydrolyze several anti-cancer and anti-inflammatory prodrugs with the vitamin B(6) pro-moiety. Finally, we evaluated antibody-catalyzed prodrug activation by examining the growth inhibition of human cervical cancer (HeLa) cells with the vitamin B(6) ester of butyric acid. These results suggest that the pro-moiety of vitamin B(6) ester is stable enough to resist natural enzymes in serum and is removed by the tailored catalytic antibodies. The combination of catalytic antibodies and prodrugs masked with vitamin B(6) would allow hydrophobic and highly toxic drugs to be used.     

单纯疱疹病毒I型SM44株胸苷激酶基因的克隆及序列测定

为了分离、克隆单纯疱疹病毒Ｉ型（ＨＳＶ－Ｉ） ＳＭ_(４４)株胸苷激酶基因（ＴＫ），采用原代兔婴肾细胞培养ＨＳＶ－ＩＳＭ_(４４) 株病毒，提取病毒核酸作为模板，设计了 ＨＳＶ－ＩＴＫ基因的上、下游引物，在引物的 ５’端分别引入 ＢａｍＨＩ和 ＥｃｏＲ Ｉ 限制性内切酶位点，通过ＰＣＲ方法扩增出 ＴＫ基因，经ＢａｍＨＩ／ＥｃｏＲＩ双酶切与 ＰＵＣ１９质粒载体相连接，构建重组 体转化受体菌ＪＭ１０９，通过筛选和酶切鉴定，获得ＨＳＶ－ＩＳＭ_(４４) ＴＫ基因阳性克隆，行全序列测定分析，与ＨＳＶ－ＩＣＬ１０１ 株 ＴＫ基因同源性为 ９９．０２％，存在 １１个核苷酸和 ７个氨基酸的差异。 ＨＳＶ－ＩＴＫ基因的克隆成功，为该基因应用于恶 性肿瘤的自杀基因治疗奠定了基础。     

Not gene therapy,but genetic surgery—the right strategy to attack cancer

In this review, I will suggest to divide all the approaches united now under common term “gene therapy” into two broad strategies of which the first one uses the methodology of targeted therapy with all its characteristics, but with genes in the role of agents targeted at a certain molecular component(s) presumably crucial for cancer maintenance. In contrast, the techniques of the other strategy are aimed at the destruction of tumors as a whole using the features shared by all cancers, for example relatively fast mitotic cell division or active angiogenesis. While the first strategy is “true” gene therapy, the second one is more like genetic surgery when a surgeon just cuts off a tumor with his scalpel and has no interest in knowing delicate mechanisms of cancer emergence and progression. I will try to substantiate the idea that the last strategy is the only right one, and its simplicity is paradoxically adequate to the super-complexity of tumors that originates from general complexity of cell regulation, strongly disturbed in tumor cells, and especially from the complexity of tumors as evolving cell populations, affecting also their ecological niche formed by neighboring normal cells and tissues. An analysis of the most widely used for such a “surgery” suicide gene/prodrug combinations will be presented in some more details.     

T cell suicide gene therapy to aid haematopoietic stem cell transplantation

Graft versus host disease (GVHD) is a T cell mediated phenomenon that arises following allogeneic haematopoietic stem cell transplantation, and may be particularly severe in the context of human leukocyte antigen (HLA) mismatched procedures. Although GVHD can be largely abrogated through T cell depletion, such measures result in loss of graft potency and reduced anti-viral and anti-leukaemic effects. The genetic modification of T cells to carry a suicide gene mechanism has been advocated as means of allowing T cells to be harnessed for their beneficial effects, and safely eliminated in the event of significant GVHD. The feasibility of the strategy has been demonstrated in clinical studies using T cells modified by retroviral transduction to encode the herpes simplex thymidine kinase (HSVTK) gene to treat patients with haematological malignancies. However, a number of limitations associated with current protocols have become apparent. Most notably, the process of retroviral transduction, which requires pre-activation of T cells, appears to impair subsequent functional potential. Efforts are now directed towards circumventing the pre-activation requirements of retroviral vectors by using alternative lentiviral systems, in association with improved suicide gene/prodrug combinations.     

Cellular suicide therapy of malignant disease

Adoptive cellular therapy is developing as a supplement or alternative to chemotherapy and/or radiation for malignant disease. Our focus is two ongoing clinical studies with transgeneic (genetically altered) cellular therapy; one uses allogeneic (from another person) lymphocytes to treat leukemia, and the second uses xenogeneic (from another species) fibroblast cells genetically altered to contain a toxin-producing suicide gene to treat ovarian cancer. Allogeneic donor lymphocyte infusions (DLI) are known to induce remission of hematologic malignancies. However, the toxicity associated with DLI is related to graft-versus-host-disease, which is due to donor lymphocytes attacking normal tissue in the recipient. Therefore, we have taken the approach of infusing DLI that have been modified to contain a latent suicide gene to treat leukemia. To treat ovarian cancer, we used xenogeneic nonimmune fibroblast-derived cells to deliver a tumor-directed cytotoxic gene to carcinoma cells. These cells release HStk transgene retroviruses that in turn transduce replicating tumor cells but not quiescent epithelium, rendering the tumor selectively susceptible to ganciclovir-mediated killing. These initial trials summarize the early stage of allogeneic/xenogeneic adoptive cellular therapy for cancer, and although the data are limited, it is encouraging to see some patients with evidence of antitumor responses. Advances in our understanding of the basic science of these treatments, together with improvements in the technology of vector design, will be required to streamline these methodologies into broader application.